Method and device for determining the concentration of heparin in a sample of fluid

ABSTRACT

A device for determining the concentration of heparin in a fluid sample comprising: a container for holding the fluid sample; a container for holding the dye solution; a mixer for mixing the fluid sample and the dye solution; an illumination source for illuminating a mixture comprising the fluid sample and the dye solution with electromagnetic radiation having a substantially continuous range of wavelengths in the visible range; a detector for detecting the absorption spectrum of the mixture within the substantially continuous range of wavelengths; a recorder for recording the absorption spectrum of the mixture within the substantially continuous range of wavelengths; and a calculator for calculating a spectral parameter.

[0001] This application is a continuation of U.S. application Ser. No.09/686,219, filed Oct. 11, 2000, the contents of which are herebyincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a method of determining theconcentration of heparin in fluid samples, and to a device forperforming the method.

BACKGROUND OF THE INVENTION

[0003] Heparin is a heteropolysaccharide of the acid mucopolysaccharidetype which has anticoagulant activity resulting from its ability tocatalyze the reaction between antithrombin III and thrombin. On thebasis of this activity, heparin is widely used as an anticoagulant incardiovascular surgery, for example, during operations withextracorporeal circulation, and in other diagnostic and therapeuticapplications.

[0004] When heparin is used as an anticoagulant, it may be very usefulto have available a method which permits rapid, reproducible andaccurate measurement of the concentration of heparin present in thesample of interest, and a device for performing this measurement.

[0005] In order to perform this measurement, it is known to use dyes ofthe cationic thiazines series. As a result of the reaction of these dyeswith heparin, it is possible to observe a change in the absorption (ortransmission) spectrum of the dye, that is, a reduction in theabsorption due to the free dye in solution and the appearance of anabsorption band of the heparin-dye complex formed. The absorptionspectrum of cationic thiazines in dilute aqueous solution is in factcharacterized by a main absorption band due to the contribution of themonomer and the dimer of the dye in solution. When heparin is added tothis dilute solution, a second band (called the μ band) whichcorresponds to the heparin-dye complex formed appears in the absorptionspectrum of the solution. The addition of heparin to the dye solutionnot only causes the appearance of the μ band, but also causes areduction in the absorption of the main band. The reduction in theabsorption of the free dye in solution and the increase in theabsorption of the dye-heparin complex constitute the two components of aphenomenon known by the term “metachromasia”, the magnitudes of which,in suitable reaction conditions, can be correlated with theconcentration of heparin present in the sample.

[0006] On the basis of this principle, assays have been developed formeasuring the concentration of heparin in samples of interest. U.S. Pat.No. 4,911,549 (Karkar) describes a method of determining the heparinconcentration in blood plasma by metachromatic reaction with the dyeAzure A which belongs to the cationic thiazines series. This assay isbased on the measurement of two distinct transmittance signals for twodistinct wavelengths, one of which is substantially insensitive to theheparin dilution.

SUMMARY OF THE INVENTION

[0007] It has surprisingly been found that the measurement of the entireabsorption spectrum of the dye in a metachromatic heparin assay, ratherthan measurement of distinct absorption signals with predeterminedseparate wavelengths, achieves results which are more accurate andreproducible and, above all, are independent of the reaction medium. Inthe case of assays performed on samples of biological fluids such asblood plasma containing proteins and other chemical species which couldpotentially interfere with accurate measurement of the absorption atthese predetermined wavelengths, this latter characteristic is ofsubstantial importance because it makes possible a device which canperform the assay automatically and which does not require repeatedcalibrations.

[0008] Accordingly, the invention provides a method for determining theconcentration of heparin in a fluid sample comprising: (a) providing afluid sample containing heparin; (b) adding to the fluid sample asolution of a dye to form a mixture of sample and dye, wherein the dyeinteracts with the heparin in the sample so that the absorption spectrumof the mixture of sample and dye in the visible range varies as a resultof the interaction in a manner quantitatively dependent on the heparinconcentration; (c) determining the absorption spectrum in the visiblerange of the mixture of sample and dye; and (d) calculating a spectralparameter representative of both the reduction in the absorption of thefree dye in solution and the increase in the absorption of thedye-heparin complex, the value of the spectral parameter beingindicative of the concentration of heparin present in the fluid sample,in order to determine the concentration of heparin present in the fluidsample. The relationship between the value of the spectral parameter andthe concentration of the heparin present in the fluid sample has beenpreviously determined by determining the absorption spectra, in thevisible range, of a composition comprising the fluid and the dye, in theabsence of heparin and in the presence of a plurality of concentrationsof heparin, and calculating the relationship between the value of thespectral parameter and the concentration of heparin. A device forperforming this method is also provided.

[0009] Additional features and advantages of the invention are set forthin the description which follows and in part will be apparent from thedescription. The objectives and other advantages of the invention willbe realized and attained by the method of determining the concentrationof heparin in a fluid sample and the device for performing this methodas particularly pointed out in the written description and claims.

[0010] It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory and are intended to provide further explanation of theinvention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the structures of thionine, Azure A, Azure B, AzureC, methylene blue, and toluidine blue, which are dyes that can be usedin the invention.

[0012]FIG. 2a shows the spectra obtained from a solution comprisingpurified Azure A and various concentrations of heparin within a range of0 to 10 IU/ml.

[0013]FIG. 2b shows a comparison between the calibration curves obtainedwith the use of the method based on the calculation of the RD parameterand with the use of purified and non-purified Azure A.

[0014]FIG. 3a shows the calibration curve obtained in Example 2.

[0015]FIG. 3b shows the change in area under the absorption spectrum ofan Azure A solution in the absence of heparin and in the presence ofheparin at concentrations of 2, 5, 7 and 10 IU/ml, respectively.

[0016]FIG. 4 shows the calibration curve obtained in Example 3.

[0017]FIG. 5 shows the calibration curve obtained in Example 4.

[0018]FIG. 6 shows a schematic of an integrated system, a device of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The invention provides a method for determining the concentrationof heparin in a fluid sample comprising: (a) providing a fluid samplecontaining heparin; (b) adding to the fluid sample a solution of a dyeto form a mixture of sample and dye, wherein the dye interacts with theheparin in the sample so that the absorption spectrum of the mixture ofsample and dye in the visible range varies as a result of theinteraction in a manner quantitatively dependent on the heparinconcentration; (c) determining the absorption spectrum in the visiblerange of the mixture of sample and dye; and (d) calculating a spectralparameter representative of both the reduction in the absorption of thefree dye in solution and the increase in the absorption of thedye-heparin complex, the value of the spectral parameter beingindicative of the concentration of heparin present in the fluid sample,in order to determine the concentration of heparin present in the fluidsample. The relationship between the value of the spectral parameter andthe concentration of the heparin present in the fluid sample has beenpreviously determined by determining the absorption spectra, in thevisible range, of a composition comprising the fluid and the dye, in theabsence of heparin and in the presence of a plurality of concentrationsof heparin, and calculating the relationship between the value of thespectral parameter and the concentration of heparin. In a preferredembodiment, the fluid sample is serum or blood plasma.

[0020] In one embodiment of the invention, the spectral parameter is thearea under the absorption spectrum in the visible range of the mixtureof sample and dye. In another embodiment of the invention, the spectralparameter is the difference in the area under the absorption spectrum inthe visible range of the mixture of sample and dye and the area underthe absorption spectrum in the visible range of a second fluid sample towhich no heparin has been added. In a preferred embodiment, the secondfluid sample to which no heparin has been added is a sample from apatient before heparin has been administered to the patient and thefluid sample is a sample from the patient after heparin has beenadministered to the patient. In still another embodiment of theinvention, the spectral parameter is a portion of the area under theabsorption spectrum in the visible range such that the variation of theportion of the area is indicative of the concentration of heparinpresent in the fluid sample.

[0021] In another embodiment of the invention, the spectral parameter isthe RD parameter, defined by the formula:

RD=A _(λ1) /A _(λ2)

[0022] in which A_(λ1) and A_(λ2) are the absorbance values measured attwo different wavelengths λ1 and λ2, respectively, and in which λ1 isselected such that the value of A_(λ1) decreases proportionally with theheparin concentration, and λ2 is selected such that the value of A_(λ2)increases proportionally with the heparin concentration.

[0023] Because the method of the present invention utilizes both of thecomponents of the metachromasia phenomenon, it achieves a high degree ofsensitivity and a low detection limit of the assay. The metachromaticassay of the present invention provides for the use of a source whichcan emit electromagnetic radiation having a substantially continuousspectrum of wavelengths in the visible range. It is thus possible bothto obtain the entire absorption spectrum of the sample of interest (thatis, the absorption values throughout the spectrum of wavelengths of theradiation emitted by the source used) and subsequently to select thewavelengths which are particularly significant in the specific assayconditions. Clearly, the selection of specific wavelengths from theentire absorption spectrum is not equivalent to the use of predeterminedspecific wavelengths.

[0024] According to one embodiment of the present invention, thevariation of the spectral parameter brought about as a result of theinteraction between heparin and dye is the variation of the area underthe absorption spectrum in the visible range of a composition comprisingthe fluid and the dye in the presence and in the absence of heparin.This variation is indicative of the concentration of heparin present inthe fluid sample. In the method according to the invention, the heparinconcentration is preferably expressed in IU/ml.

[0025] The term “area” is intended to define either the area under eachabsorption spectrum in the entire visible range, or a portion of thisarea selected in a manner such that it is representative of bothcomponents of the metachromasia phenomenon and its variation is thusindicative of the concentration of heparin present in the fluid sample.

[0026] Equivalent performance of the test according to the invention canbe achieved by measuring the variation in the presence and in theabsence of heparin of another spectral parameter which is alsoindicative of the concentration of heparin present in the fluid sample,that is, the variation of the RD parameter, defined as:

RD=A _(λ1) /A _(λ2)

[0027] in which A_(λ1) and A_(λ2) are the absorption values measured atwavelengths λ1 and λ2, respectively and in which λ1 is selected suchthat the value of A_(λ1) decreases proportionally with the heparinconcentration whereas λ2 is selected such that the value of A_(λ2)increases proportionally with the heparin concentration. In other words,within the absorption spectrum in the visible range of the compositioncomprising the fluid and the dye, two wavelengths (λ1 and λ2) areselected such that each is representative of one of the two componentsof the metachromasia phenomenon defined above.

[0028] The values of λ1 and λ2 selected from the absorption spectrum aresuch as to be optimal in relation to the dye used and to the specificassay conditions. Preferred values of λ1 and λ2 fall, for example,within the ranges of 560 to 610 nm and 480 to 530 nm, respectively;within these wavelength ranges, for example, 590 nm and 510 nm,respectively, are more preferred.

[0029] A method as described above in which transmittance measurementsare performed also falls within the scope of the present invention sincetransmittance (T) is linked to absorbance (A) by the following equation:

A=log₁₀ (1/T).

[0030] The dye solution can comprise a non-ionic surfactant. Theconcentration of the dye present in the solution is preferably withinthe range of 1×10⁻³ to 1×10⁻⁶ moles/liter, more preferably about 5×10⁻⁵moles/liter. The ionic strength of the dye solution is preferably lessthan 0.1. Moreover, the dye solution is preferably added to the fluidsample in a proportion of from 5:1 to 100:1 by volume, according to thedye used and the dynamic range required. More preferably, the proportionis 10:1.

[0031] The dye used in the metachromatic assay of the invention ispreferably a cationic thiazine selected from thionine, Azure A, Azure B,Azure C, methylene blue, toluidine blue and mixtures thereof, preferablyof known composition. The structures of these molecules are shown inFIG. 1.

[0032] The use of Azure A is more preferred, even more preferably with adegree of purity of from 90 to 100%, and still more preferably with adegree of purity of 95 to 99%. In embodiments of the invention, theconcentration of Azure A in the dye solution is within the range of from1×10⁻³ to 1×10⁻⁶ moles/liter, more preferably about 5×10⁻⁵ moles/liter.In other embodiments of the invention, the dye is Azure A and λ1 isselected within the range of from 560 to 610 nm and λ2 is selectedwithin the range of from 480 to 530 nm, more preferably, λ1 is 590 nmand λ2 is 510 nm.

[0033] As reported in U.S. Pat. No. 4,003,892 (Löhr et al.), commercialcationic thiazines of the methylene blue homologous series are in factactually compositions comprising at least two members of the groupconsisting of thionine, Azure A, Azure B, Azure C and methylene blue invariable proportions. Each of these dyes is in fact produced byoxidative demethylation starting with methylene blue (the molecule ofthe series with the highest degree of N-methylation) which produces acomplex mixture comprising the molecule desired and other thiazines ofthe same series in variable proportions.

[0034] These dyes of the methylene blue homologous series have beenpurified by chromatography in silica-gel columns with the use of amixture of water, acetic acid and formic acid as an eluent, as describedin the patent mentioned above and in greater detail in Example 1.

[0035] Each of the five fractions thus obtained (each of whichrepresents a single cationic thiazine) was further subjected to reversephase HPLC chromatography with the use of a decreasing polarity gradientin order to evaluate its degree of purity. In the case of Azure A, adegree of purity greater than 90% was assessed. In the method accordingto the invention, the use of Azure A with a degree of purity of from 95to 99% is therefore even more preferred.

[0036] If the spectrum of the purified Azure A dye is analyzed, theappearance of a new absorption band with a maximum at about 590 nm isobserved. This band, known as the β band, is due to the absorptionsolely of the dimer of the dye in solution. Moreover, in the purifiedAzure A spectrum, the maximum absorption band at 630 nm (the α band)which is already visible in the spectrum of the non-purified dye, inwhich it was indicated as the main band, is considerably narrower. Inthe spectrum of the purified dye, the contributions of the dimer (βband) and of the monomer (α band) of the dye can thus be clearlydistinguished. If the spectra of the purified Azure A dye in thepresence of heparin are analyzed, it becomes clear that the regioncorresponding to the β band is also involved in the metachromasiaphenomenon, since the absorbance within this region varies in a mannerquantitatively dependent on the concentration of heparin present in thefluid sample. FIG. 2a shows the spectra obtained from a solutioncomprising purified Azure A and various concentrations of heparin withina range of 0 to 10 IU/ml.

[0037] The use of the Azure A dye purified as described above and ingreater detail in Example 1 below increases the sensitivity of theassay. FIG. 2b is a comparison between the calibration curves obtainedwith the use of the method based on the calculation of the RD parameterand with the use of purified and non-purified Azure A. The steeper slopeof the curve obtained with the use of purified Azure A illustrates theincrease in sensitivity obtained for given assay conditions.

[0038] The invention provides a device for determining the concentrationof heparin in a fluid sample, comprising:

[0039] a container for holding the fluid sample;

[0040] a container for holding the dye solution;

[0041] a mixer for mixing the fluid sample and the dye solution;

[0042] an illumination source for illuminating a mixture comprising thefluid sample and the dye solution with electromagnetic radiation havinga substantially continuous range of wavelengths in the visible range;

[0043] a detector for detecting the absorption spectrum of the mixturewithin the substantially continuous range of wavelengths;

[0044] a recorder for recording the absorption spectrum of the mixturewithin the substantially continuous range of wavelengths; and

[0045] a calculator for calculating a spectral parameter;

[0046] wherein the value of the spectral parameter is indicative of theconcentration of heparin present in the fluid sample, and the spectralparameter is representative of both the reduction in the absorption ofthe free dye in solution and the increase in the absorption of thedye-heparin complex in a method in which the dye solution is added tothe fluid sample to form a mixture of sample and dye, and the dyeinteracts with the heparin in the sample so that the absorption spectrumof the mixture of sample and dye in the visible range varies as a resultof the interaction in a manner quantitatively dependent on the heparinconcentration.

[0047] In an embodiment of the invention, the illumination source forilluminating the composition comprises at least one light-emitting diodehaving substantially continuous multiple wavelengths in the visiblerange. In another embodiment, the illumination source further comprisesat least one diode emitting light in the red region (for example,between 620 and 750 nm).

[0048] In an embodiment of the invention, the container for holding thefluid sample comprises a hollow fiber filter with a porosity to separatethe corpuscular portion of blood from the plasma. In another embodiment,the device further comprises a holder for holding a detergent solutionand a holder for holding a waste solution. The device can furthercomprise a computer.

[0049] The following examples are provided by way of illustration butare not intended to limit the scope of the present invention in any way.

EXAMPLE 1 Purification of Azure A Dye

[0050] The purification of the components of the methylene bluehomologous series was performed with the use of the method described byLöhr et al, in U.S. Pat. No. 4,003,892, modified so as to improve it,particularly in terms of yield and recovery of the eluent.

[0051] 2.5 g of commercial Azure A (Aldrich) dissolved in 75 ml ofeluent (10% acetic acid and 5% formic acid in aqueous solution) wassupplied to a Merck 60 240-400 mesh silica-gel column. The dimensions ofthe column were 50 mm diameter and 1000 mm height. The flow was 6ml/min.

[0052] In these conditions, six fractions, each corresponding to a dyein the methylene blue homologous series were separated: Fraction 1Bernthsen violet A = 580 nm Fraction 2 Thionine A = 600 nm Fraction 3Azure C A = 613 nm Fraction 4 Azure A A = 630 nm Fraction 5 Azure B A =650 nm Fraction 6 Methylene blue A = 670 nm

[0053] Fraction 4 (Azure A) was diverted and adsorbed in a Supelco XAD 2column with a flow of 1 ml/min. The dimensions of the column were 40 mmdiameter and 350 mm height. During this adsorption stage in particularit is preferable to avoid light as much as possible; this was achievedby covering the apparatus and particularly the columns with aluminiumfoil.

[0054] After the Azure A had been fully adsorbed, the column was washedwith 1 litre of a 5% NaCl solution so as to exchange the organic anionswith Cl⁻. The column was then washed with distilled water until anegative chlorides reaction (reaction with AgNO₃) was obtained. Thiswashing was performed with a counterflow to prevent loss of dye. Afterthe washing, the Azure A dye was eluted with pure methanol. Themethanolic solution of Azure A was evaporated to approximately 10 ml,filtered and added dropwise to an excess of ether (about 500 ml) fromwhich the solid Azure A precipitated. The yield of the method was about50% by weight.

[0055] The silica-gel column can be regenerated by washing withdistilled water, a 3% solution of potassium permanganate in water,followed by a 3% sodium dithionite, sulphite or metabisulphite solution,distilled water again and, finally, re-equilibration with the 10% aceticacid/5% formic acid solution.

[0056] It is also possible to regenerate 70-80% of the eluent by passingthe fractions containing the dyes, except for the Azure A dye, through acolumn of activated carbon (30-250 mm) which captures the dyes, leavingthe eluent clean.

EXAMPLE 2 Metachromatic Assay for Heparin with the Use of Purified AzureA Dye, Areas Method

[0057] 1 ml of a 5×10⁻⁵ moles/liter solution, in deionized water wateralso including 1% v/v of a non-ionic surfactant (Pluronic F68, apolyoxyalkylene made by BASF Aktiengesellschaft, Ludwigshaven, Germany),of Azure A (Aldrich) purified in accordance with Example 1 was added toa 100 μl sample of horse serum (Sigma) containing an unknownconcentration of added sodium heparin (Liquemin, Roche) expressed inIU/ml.

[0058] The composition thus produced was mixed for a few seconds andtransferred to an optical cuvette for the detection of the absorptionspectrum. For this purpose, a Perkins Elmer spectrophotometer modelLambda 2 with a tungsten lamp was used. The spectrophotometer wasconnected to a personal computer for acquiring and processing the data.The area under the absorption spectrum of the sample between 400 nm and800 nm was calculated by integration.

[0059] The same method was repeated with a composition comprising 100 μlof the same serum without added heparin, and 1 ml of a dye solution asdefined above.

[0060] The change in the area under the two absorption spectra obtainedwas calculated and this numeric value was used to calculate theconcentration of heparin present in the serum sample with the use of acalibration curve constructed beforehand using serum samples containingdifferent known heparin concentrations. FIG. 3a shows the calibrationcurve obtained. FIG. 3b comprises four graphs showing the change in thearea under the absorption spectrum of an Azure A solution in the absenceof heparin and in the presence of heparin at concentrations of 2, 5, 7and 10 IU/ml, respectively.

[0061] The detection limit of the assay, defined as the concentration ofanalyte which produces a signal of an intensity equal to 2.58 σ₀ (inwhich σ₀ is the standard deviation of the signal obtained in the absenceof heparin) was 0.1 IU/ml. Within the heparin concentration range offrom 0 IU/ml to 10 IU/ml, a sensitivity of the assay of 3.44 (units ofarea)×(IU/ml)⁻¹ was calculated.

EXAMPLE 3 Metachromatic Assay for Heparin with the Use of Purified AzureA Dye, Method Based on Calculation of the RD Parameter

[0062] Azure A dye purified as described in Example 1 was used todetermine the concentration of heparin in a fluid sample (serum) withthe use of the method based on the calculation of the RD parameter asdefined above. Values of λ1 and λ2 which had been found to be optimalfor the specific assay conditions, that is 590 nm and 510 nm,respectively, were used. The value of RD was thus calculated by theformula:

RD=A _(590nm) /A _(510nm)

[0063] Both of the values of A_(590 nm) and A_(510 nm) were alsocorrected for the absorbance value at the base line of the spectrum(A_(800 nm)). FIG. 4 shows the calibration curve obtained. The detectionlimit of the assay was 0.1 IU/ml. The sensitivity of the assay,calculated within the range of heparin concentration of from 0 IU/ml to3 IU/ml, was 2.54 (Units of RD)×(IU/ml)⁻¹.

EXAMPLE 4 Metachromatic Assay for Heparin with the Use of Toluidine BlueAreas Method

[0064] The metachromatic assay according to the method described inExample 2 was performed with the use of the commercial dye toluidineblue (Aldrich). The assay conditions were those described in Example 2above, except that the determination was performed on a sample of bovineplasma. FIG. 5 shows the calibration curve obtained. The sensitivity ofthe metachromatic assay according to this embodiment within the range ofheparin concentrations of from 0 IU/ml to 10 IU/ml was 1.19 (Units ofarea)×(IU/ml)⁻¹.

EXAMPLE 5 Integrated System

[0065]FIG. 6 shows an embodiment of a device according to the invention.This device, known as an “integrated system” is particularly suitablefor performing the metachromatic assay for heparin according to theinvention on a sample of blood plasma from a patient undergoing asurgical operation with extracorporeal circulation. The device isconstituted by four separate sections:

[0066] (1) Blood treatment section: constituted by a filter, forexample, a filter of hollow polypropylene fibres with a pore diameter of0.55 μm and an effective filtration area of 0.1 m² (Hemaplex X® BT 900,Dideco), connected in parallel with a circuit for extracorporealcirculation. This filter separates the corpuscular portion of the bloodfrom the plasma without interfering with the heparin concentrationpresent in the plasma.

[0067] (2) Metering section: constituted by a peristaltic pump (P1) withtwo channels and two pinch valves (V1 and V2). The tubes used were ofTygon® with the following cross-sections: ID {fraction (3/32)}″, OD{fraction (5/32)}″, WALL {fraction (1/32)}″ for the dye, and ID{fraction (1/32)}″, OD {fraction (3/32)}″, WALL {fraction (1/32)}″ forthe plasma. This section has the function of metering both the plasmacoming from the filter and the dye solution in order to produce acomposition comprising plasma and dye, and also of admitting a detergentsolution into the device to clean the device when necessary.

[0068] (3) Measurement section: constituted by a cuvette-holder, acuvette, a source of electromagnetic radiation in the visible range, andan optical-fibre spectrophotometer. The holder is constructed forhousing the cuvette, engaging the source of electromagnetic radiationand of the optical fibre spectrophotometer, and collimating the beam ofelectromagnetic radiation in the cuvette.

[0069] (4) Liquids section: constituted by a container for the dyesolution, a container for the detergent solution and a container forcollecting the waste solutions.

[0070] The metering section of the device is controlled electronically,preferably by two electronic cards. The device is also connected to apersonal computer for the control of the metering section and theacquisition and processing of the data coming from thespectrophotometer.

[0071] The above description and accompanying drawings are provided forthe purpose of describing embodiments of the invention and are notintended to limit the scope of the invention in any way. It will beapparent to those skilled in the art that various modifications andvariations can be made in the method for determining the concentrationof heparin in a fluid sample and the device for performing this methodwithout departing from the spirit or scope of the invention. Thus, it isintended that the present invention cover the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

What is claimed is:
 1. A device for determining the concentration ofheparin in a fluid sample, comprising: a container for holding the fluidsample; a container for holding the dye solution; a mixer for mixing thefluid sample and the dye solution; an illumination source forilluminating a mixture comprising the fluid sample and the dye solutionwith electromagnetic radiation having a substantially continuous rangeof wavelengths in the visible range; a detector for detecting theabsorption spectrum of the mixture within the substantially continuousrange of wavelengths; a recorder for recording the absorption spectrumof the mixture within the substantially continuous range of wavelengths;and a calculator for calculating a spectral parameter; wherein the valueof the spectral parameter is indicative of the concentration of heparinpresent in the fluid sample, and the spectral parameter isrepresentative of both the reduction in the absorption of the free dyein solution and the increase in the absorption of the dye-heparincomplex in a method in which the dye solution is added to the fluidsample to form a mixture of sample and dye, and the dye interacts withthe heparin in the sample so that the absorption spectrum of the mixtureof sample and dye in the visible range varies as a result of theinteraction in a manner quantitatively dependent on the heparinconcentration.
 2. A device according to claim 1, wherein theillumination source for illuminating the composition comprises at leastone light-emitting diode having substantially continuous multiplewavelengths in the visible range.
 3. A device according to claim 2,wherein the illumination source further comprises at least one diodeemitting light in the red region.
 4. A device according to claim 1,wherein the container for holding the fluid sample comprises a hollowfiber filter with a porosity to separate the corpuscular portion ofblood from the plasma.
 5. A device according to claim 4, wherein thedevice further comprises a holder for holding a detergent solution and aholder for holding a waste solution.
 6. A device according to claim 1,wherein the device further comprises a computer.